A potential pathway for flippase-facilitated glucosylceramide catabolism in plants.

The Aminophospholipid ATPase (ALA) family of plant lipid flippases is involved in the selective transport of lipids across membrane bilayers. Recently, we demonstrated that double mutants lacking both ALA4 and -5 are severely dwarfed. Dwarfism in ala4/5 mutants was accompanied by cellular elongation defects and various lipidomic perturbations, including a 1.4-fold increase in the accumulation of glucosylceramides (GlcCers) relative to total sphingolipid content. Here, we present a potential model for flippase-facilitated GlcCer catabolism in plants, where a combination of ALA flippases transport GlcCers to cytosolic membrane surfaces where they are degraded by Glucosylceramidases (GCDs). GCDs remove the glucose headgroup from GlcCers to produce a ceramide (Cer) backbone, which can be further degraded to sphingoid bases (Sphs, e.g, phytosphingosine) and fatty acids (FAs). In the absence of GlcCer-transporting flippases, GlcCers are proposed to accumulate on extracytoplasmic (i.e., apoplastic) or lumenal membrane surfaces. As GlcCers are potential precursors for Sph production, impaired GlcCer catabolism might also result in the decreased production of the secondary messenger Sph-1-phosphate (Sph-1-P, e.g., phytosphingosine-1-P), a regulator of cell turgor. Importantly, we postulate that either GlcCer accumulation or reduced Sph-1-P signaling might contribute to the growth reductions observed in ala4/5 mutants. Similar catabolic pathways have been proposed for humans and yeast, suggesting flippase-facilitated GlcCer catabolism is conserved across eukaryotes.

Mutation of a bHLH transcription factor allowed almond domestication.

Wild almond species accumulate the bitter and toxic cyanogenic diglucoside amygdalin. Almond domestication was enabled by the selection of genotypes harboring sweet kernels. We report the completion of the almond reference genome. Map-based cloning using an F1 population segregating for kernel taste led to the identification of a 46-kilobase gene cluster encoding five basic helix-loop-helix transcription factors, bHLH1 to bHLH5. Functional characterization demonstrated that bHLH2 controls transcription of the P450 monooxygenase-encoding genes PdCYP79D16 and PdCYP71AN24, which are involved in the amygdalin biosynthetic pathway. A nonsynonymous point mutation (Leu to Phe) in the dimerization domain of bHLH2 prevents transcription of the two cytochrome P450 genes, resulting in the sweet kernel trait.

Phospholipid flipping involves a central cavity in P4 ATPases.

P4 ATPase flippases translocate phospholipids across biomembranes, thus contributing to the establishment of transmembrane lipid asymmetry, a feature important for multiple cellular processes. The mechanism by which such phospholipid flipping occurs remains elusive as P4 ATPases transport a giant substrate very different from that of other P-type ATPases such as Na+/K+- and Ca2+-ATPases. Based on available crystal structures of cation-transporting P-type ATPases, we generated a structural model of the broad-specificity flippase ALA10. In this model, a cavity delimited by transmembrane segments TM3, TM4, and TM5 is present in the transmembrane domain at a similar position as the cation-binding region in related P-type ATPases. Docking of a phosphatidylcholine headgroup in silico showed that the cavity can accommodate a phospholipid headgroup, likely leaving the fatty acid tails in contact with the hydrophobic portion of the lipid bilayer. Mutagenesis data support this interpretation and suggests that two residues in TM4 (Y374 and F375) are important for coordination of the phospholipid headgroup. Our results point to a general mechanism of lipid translocation by P4 ATPases, which closely resembles that of cation-transporting pumps, through coordination of the hydrophilic portion of the substrate in a central membrane cavity.

Heterologous expression and purification of membrane-bound pyrophosphatases.

Membrane-bound pyrophosphatases (M-PPases) are enzymes that couple the hydrolysis of inorganic pyrophosphate to pumping of protons or sodium ions. In plants and bacteria they are important for relieving stress caused by low energy levels during anoxia, drought, nutrient deficiency, cold and low light intensity. While they are completely absent in mammalians, they are key players in the survival of disease-causing protozoans making these proteins attractive pharmacological targets. In this work, we aimed at the purification of M-PPases in amounts suitable for crystallization as a first step to obtain structural information for drug design. We have tested the expression of eight integral membrane pyrophosphatases in Saccharomyces cerevisiae, six from bacterial and archaeal sources and two from protozoa. Two proteins originating from hyperthermophilic organisms were purified in dimeric and monodisperse active states. To generate M-PPases with an increased hydrophilic surface area, which potentially should facilitate formation of crystal contacts, phage T4 lysozyme was inserted into different extramembraneous loops of one of these M-PPases. Two of these fusion proteins were active and expressed at levels that would allow their purification for crystallization purposes.

Flippases: still more questions than answers.

Our understanding of flippase-mediated lipid translocation and membrane vesiculation, and the involvement of P-type ATPases in these processes is just beginning to emerge. The results obtained so far demonstrate significant complexity within this field and point to major tasks for future research. Most importantly, biochemical characterization of P(4)-ATPases is required in order to clarify whether these transporters indeed are capable of catalyzing transmembrane phospholipid flipping. The beta-subunit of P(4)-ATPases shows unexpected similarities between the beta- and gamma-subunits of the Na+/K+-ATPase. It is likely that these proteins provide a similar solution to similar problems, and might have adopted similar structures to accomplish these tasks. No P(4)-ATPases have been identified in the endoplasmic reticulum and it remains an intriguing possibility that, in this compartment, P(5A)-ATPases are functional homologues of P(4)-ATPases.

A thermostable K(+)-stimulated vacuolar-type pyrophosphatase from the hyperthermophilic bacterium Thermotoga maritima.

Current evidence suggests the occurrence of two classes of vacuolar-type H(+)-translocating inorganic pyrophosphatases (V-PPases): K(+)-insensitive proteins, identified in eukaryotes, bacteria and archaea, and K(+)-stimulated V-PPases, identified to date only in eukaryotes. Here, we describe the functional characterization of a thermostable V-PPase from the anaerobic hyperthermophilic bacterium Thermotoga maritima by heterologous expression in Saccharomyces cerevisiae. The activity of this 71-kDa membrane-embedded polypeptide has a near obligate requirement for K(+), like the plant V-PPase, and its thermostability depends on the binding of Mg(2+). Phylogenetic analysis of protein sequences consistently assigned the T. maritima V-PPase to the K(+)-sensitive class of V-PPases so far only known for eukaryotes. The finding of a K(+)-stimulated V-PPase also in a member of a primitive eubacterial lineage strongly supports an ancient evolutionary origin of this group of pyrophosphate-energized proton pumps.

Enzymatic systems of inorganic pyrophosphate bioenergetics in photosynthetic and heterotrophic protists: remnants or metabolic cornerstones?

An increasing body of biochemical and genetic evidence suggests that inorganic pyrophosphate (PPi) plays an important role in protist bioenergetics. In these organisms, two types of inorganic pyrophosphatases [EC 3.6.1.1, namely soluble PPases (sPPases) and proton-translocating PPases (H+-PPases)] that hydrolyse the PPi generated by cell anabolism, thereby replenishing the orthophosphate pool needed for phosphorylation reactions, are present in different cellular compartments. Photosynthetic and heterotrophic protists possess sPPases located in cellular organelles (plastids and mitochondria), where many anabolic and biosynthetic reactions take place, in addition to H+-PPases, which are integral membrane proteins of the vacuolysosomal membranes and use the chemical energy of PPi to generate an electrochemical proton gradient useful in cell bioenergetics. This last category of proton pumps was considered to be restricted to higher plants and some primitive photosynthetic bacteria, but it has been found recently in many protists (microalgae and protozoa) and bacteria, thus indicating that H+-PPases are much more widespread than previously thought. No cytosolic sPPase (in bacteria, fungi and animal cells) has been shown to occur in these lower eukaryotes. The widespread occurrence of these key enzymes of PPi metabolism among evolutionarily divergent protists strongly supports the ancestral character of the bioenergetics based on this simple energy-rich compound, which may play an important role in survival under different biotic and abiotic stress conditions.